Methods and devices for cellular activation

ABSTRACT

An apparatus includes multiple first reservoirs and multiple second reservoirs joined with a substrate. Selected ones of the multiple first reservoirs include a reducing agent, and first reservoir surfaces of selected ones of the multiple first reservoirs are proximate to a first substrate surface. Selected ones of the multiple second reservoirs include an oxidizing agent, and second reservoir surfaces of selected ones of the multiple second reservoirs are proximate to the first substrate surface.

The present application claims priority to U.S. Provisional PatentApplication Numbers 61/818,797 filed May 2, 2013, 61/821,362, filed May9, 2013, and 61/821,365, filed May 9, 2013, each of which areincorporated by reference herein in their entireties.

FIELD

Biologic tissues and cells, microbes, bacteria, viruses, fungi, andother organisms or organic matter can be affected by electricalstimulus. Accordingly, apparatus and techniques for applying electricstimulus to organic matter have been developed to address a number ofmedical issues. The present specification relates to methods and devicesuseful for directing cell migration, increasing cell nutrient uptake,promoting wound healing, reducing inflammation, and providingantibacterial effects.

SUMMARY

Aspects disclosed herein comprise bioelectric devices that comprise amulti-array matrix of biocompatible microcells. Such matrices caninclude a first array comprising a pattern of microcells formed from afirst conductive solution, the solution including a metal species; and asecond array comprising a pattern of microcells formed from a secondconductive solution, the solution including a metal species capable ofdefining at least one voltaic cell for spontaneously generating at leastone electrical current with the metal species of the first array whensaid first and second arrays are introduced to an electrolytic solutionand said first and second arrays are not in physical contact with eachother. Certain aspects utilize an external power source such as AC or DCpower or pulsed RF or pulsed current, such as high voltage pulsedcurrent. In one embodiment, the electrical energy is derived from thedissimilar metals creating a battery at each cell/cell interface,whereas those embodiments with an external power source may requireconductive electrodes in a spaced apart configuration to predeterminethe electric field shape and strength. The external source could provideenergy for a longer period than the batteries on the surface.

The devices can also generate a localized electric field in a patterndetermined by the distance and physical orientation of the cells orelectrodes. Effective depth of the electric field can be predeterminedby the orientation and distance of the cells or electrodes. In aspectsthe devices can be coated either totally or partially with a hydrogel,or glucose or any other drug, cellular nutrition, stem cells, or otherbiologic. In embodiments the electric field can be extended, for examplethrough the use of a hydrogel. In certain embodiments, for exampletreatment methods, it can be preferable to utilize AC or DC current.

Further aspects include a method of directing cell migration using adevice disclosed herein. These aspects include methods of improvingre-epithelialization.

Further aspects include methods of increasing glucose uptake as well asmethods of increasing cellular thiol levels. Additional aspects includea method of energizing mitochondria.

Further aspects include a method of stimulating cellular proteinexpression.

Further aspects include a method of stimulating cellular DNA synthesis.

Further aspects include a method of stimulating cellular Ca²⁺ uptake.

Aspects of the invention include devices and methods for increasingcapillary density.

Embodiments include devices and methods for increasing transcutaneouspartial pressure of oxygen. Further embodiments include methods anddevices for treating or preventing pressure ulcers.

Additional aspects include a method of preventing bacterial biofilmformation. Aspects also include a method of reducing microbial orbacterial proliferation, killing microbes or bacteria, killing bacteriathrough a biofilm layer, or preventing the formation of a biofilm.Embodiments include methods using devices disclosed herein incombination with antibiotics for reducing microbial or bacterialproliferation, killing microbes or bacteria, killing bacteria through abiofilm layer, or preventing the formation of a biofilm.

Further aspects include methods of treating diseases related tometabolic deficiencies, such as diabetes, or other disease wherein thepatient exhibits a compromised metabolic status.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a detailed plan view of an embodiment disclosed herein.

FIG. 2 is a detailed plan view of a pattern of applied electricalconductors in accordance with an embodiment disclosed herein.

FIG. 3 is an adhesive bandage using the applied pattern of FIG. 2.

FIG. 4 is a cross-section of FIG. 3 through line 3-3.

FIG. 5 is a detailed plan view of an alternate embodiment disclosedherein which includes fine lines of conductive metal solution connectingelectrodes.

FIG. 6 is a detailed plan view of another alternate embodiment having aline pattern and dot pattern.

FIG. 7 is a detailed plan view of yet another alternate embodimenthaving two line patterns.

FIG. 8A depicts an embodiment showing the location of discontinuousregions as well as anchor regions of the system.

FIG. 8B depicts an embodiment showing the location of discontinuousregions as well as anchor regions of the system.

FIG. 8C depicts an embodiment showing the location of discontinuousregions as well as anchor regions of the system.

FIG. 8D depicts an embodiment showing the location of discontinuousregions as well as anchor regions of the system.

FIG. 8E depicts an embodiment showing the location of discontinuousregions as well as anchor regions of the system.

FIG. 9A depicts an embodiment with two discrete circuits (92 and 94).

FIG. 9B depicts an embodiment with power terminals (96 and 98) for usewith an external power source.

FIG. 10 depicts a disclosed embodiment including slits (102).

DETAILED DESCRIPTION

Embodiments disclosed herein include systems that can provide a lowlevel electric field (LLEF) to a tissue or organism (thus a “LLEFsystem”) or, when brought into contact with an electrically conductingmaterial, can provide a low level micro-current (LLMC) to a tissue ororganism (thus a “LLMC system”). Thus, in embodiments a LLMC system is aLLEF system that is in contact with an electrically conducting material.In certain embodiments, the micro-current or electric field can bemodulated, for example, to alter the duration, size, shape, field depth,current, polarity, or voltage of the system. In embodiments thewatt-density of the system can be modulated.

Embodiments disclosed herein comprise patterns of microcells. Thepatterns can be designed to produce an electric field, an electriccurrent, or both over living cells. In embodiments the pattern can bedesigned to produce a specific size, strength, density, shape, orduration of electric field or electric current. In embodiments reservoiror dot size and separation can be altered.

In embodiments devices disclosed herein can apply an electric field, anelectric current, or both wherein the field, current, or both can be ofvarying size, strength, density, shape, or duration in different areasof a wound or tissue. In embodiments, by micro-sizing the electrodes orreservoirs, the shapes of the electric field, electric current, or bothcan be customized, increasing or decreasing very localized wattdensities and allowing for the design of “smart patterned electrodes”where the amount of e field over a tissue can be designed or produced oradjusted based on feedback from the tissue or on an algorithm within thesensors and fed-back to a control module. The electric field, electriccurrent, or both can be strong in one zone and weaker in another. Theelectric field, electric current, or both can change with time and bemodulated based on treatment goals or feedback from the tissue orpatient. The control module can monitor and adjust the size, strength,density, shape, or duration of electric field or electric current basedon tissue parameters.

A dressing disclosed herein and placed over tissue such as a joint inmotion can move relative to the tissue. Reducing the amount of motionbetween tissue and dressing can be advantages to healing. Inembodiments, traction or friction blisters can be treated, minimized, orprevented. Slotting or placing strategic cuts into the dressing can makeless friction on the wound. In embodiments, use of an elastic dressingsimilar to the elasticity of the skin is also possible. The use of thedressing as a temporary bridge to reduce stress across the wound sitecan reduce stress at the sutures or staples and this will reducescarring and encourage healing.

The devices can be used to modulate cell characteristics, such as forexample to direct and promote cell migration or infiltration, or toincrease uptake of materials such as glucose, or to increase cellsignaling activity or to defeat bacterial signaling such as quorumsensing. The devices can be used therapeutically, such as to promote thehealing of wounds, or in the treatment of disease such as those relatedto metabolic deficiencies, such as diabetes. Further disclosure relatingto the use of electrical current to heal wounds can be found in U.S.Pat. No. 7,457,667 entitled CURRENT PRODUCING SURFACE FOR A WOUNDDRESSING issued Nov. 25, 2008, which is incorporated herein by referencein its entirety.

Embodiments disclosed herein comprise biocompatible electrodes orreservoirs or dots on a surface, for example a fabric or the like. Inembodiments the surface can be pliable. In embodiments the surface cancomprise a gauze or mesh. Suitable types of pliable surfaces for use inembodiments disclosed herein can be absorbent textiles, low-adhesives,vapor permeable films, hydrocolloids, hydrogels, alginates, foams,foam-based materials, cellulose-based materials including Kettenbachfibers, hollow tubes, fibrous materials, such as those impregnated withanhydrous/hygroscopic materials, beads and the like, or any suitablematerial as known in the art. In embodiments the pliable material canform, for example, a bandage, a wrist band, a neck band, a waist band, awound dressing, cloth, fabric, or the like. Embodiments can includecoatings on the surface, such as, for example, over or between theelectrodes. Such coatings can include, for example, silicone, andelectrolytic mixture, hypoallergenic agents, drugs, biologics, stemcells, skin substitutes or the like. Drugs suitable for use withembodiments of the invention include analgesics, antibiotics,anti-inflammatories, or the like. In embodiments the electric field orcurrent produced can “drive” the drug through the skin or surfacetissue.

In embodiments the material can include a port to access the interior ofthe material, for example to add fluid, gel, or some other material tothe dressing. Certain embodiments can comprise a “blister” top that canenclose a material. In embodiments the blister top can contain amaterial that is released into the dressing when the blister is pressed,for example a liquid.

In embodiments the system comprises a component such as elastic tomaintain or help maintain its position. In embodiments the systemcomprises a component such as an adhesive to maintain or help maintainits position. The adhesive component can be covered with a protectivelayer that is removed to expose the adhesive at the time of use. Inembodiments the adhesive can comprise, for example, sealants, such ashypoallergenic sealants, gecko sealants, mussel sealants, waterproofsealants such as epoxies, and the like.

In embodiments the positioning component can comprise an elastic filmwith an elasticity, for example, similar to that of skin, or greaterthan that of skin, or less than that of skin. In embodiments, the LLMCor LLEF system can comprise a laminate where layers of the laminate canbe of varying elasticities. For example, an outer layer may be highlyelastic and an inner layers in-elastic. The in-elastic layer can be madeto stretch by placing stress relieving discontinuous regions or slitsthrough the thickness of the material so there is a mechanicaldisplacement rather than stress that would break the fabric weave beforestretching would occur. In embodiments the slits can extend completelythrough a layer or the system or can be placed where expansion isrequired. In embodiments of the system the slits do not extend all theway through the system or a portion of the system such as the dressingmaterial. In embodiments the discontinuous regions can pass halfwaythrough the long axis of the wound management system.

In certain embodiments the surface can comprise the surface of, forexample, a catheter, or a microparticle. Such embodiments can be used totreat a subject internally both locally or systemically. For example,the microparticles can be used to make a pharmaceutical composition incombination with a suitable carrier. In embodiments nanotechnology suchas nanobots can be used to provide LLMC systems that can be used ascomponents of pharmaceutical formulations, such as injected, inhaled, ororally administered formulations.

“Activation gel” as used herein means a composition useful formaintaining a moist environment about the wound or promoting healingwithin and about the wound.

“Affixing” as used herein can mean contacting a patient or tissue with adevice or system disclosed herein.

“Applied” or “apply” as used herein refers to contacting a surface witha conductive material, for example printing, painting, or spraying aconductive ink on a surface. Alternatively, “applying” can meancontacting a patient or tissue or organism with a device or systemdisclosed herein.

“Cell infiltration” as used herein refers to cell migration to a targettissue or area to which cell migration is desired, for example a wound.

“Conductive material” as used herein refers to an object or type ofmaterial which permits the flow of electric charges in one or moredirections. Conductive materials can include solids such as metals orcarbon, or liquids such as conductive metal solutions and conductivegels. Conductive materials can be applied to form at least one matrix.Conductive liquids can dry, cure, or harden after application to form asolid material.

“Discontinuous region” as used herein refers to a “void” in a materialsuch as a hole, slot, or the like. The term can mean any void in thematerial though typically the void is of a regular shape. The void inthe material can be entirely within the perimeter of a material or itcan extend to the perimeter of a material.

“Dots” as used herein refers to discrete deposits of dissimilarreservoirs that can function as at least one battery cell. The term canrefer to a deposit of any suitable size or shape, such as squares,circles, triangles, lines, etc. The term can be used synonymously with,microcells, etc.

“Electrode” refers to similar or dissimilar conductive materials. Inembodiments utilizing an external power source the electrodes cancomprise similar conductive materials. In embodiments that do not use anexternal power source, the electrodes can comprise dissimilar conductivematerials that can define an anode and a cathode.

“Expandable” as used herein refers to the ability to stretch whileretaining structural integrity and not tearing. The term can refer tosolid regions as well as discontinuous or void regions; solid regions aswell as void regions can stretch or expand.

“Galvanic cell” as used herein refers to an electrochemical cell with apositive cell potential, which can allow chemical energy to be convertedinto electrical energy. More particularly, a galvanic cell can include afirst reservoir serving as an anode and a second, dissimilar reservoirserving as a cathode. Each galvanic cell can store chemical potentialenergy. When a conductive material is located proximate to a cell suchthat the material can provide electrical and/or ionic communicationbetween the cell elements the chemical potential energy can be releasedas electrical energy. Accordingly, each set of adjacent, dissimilarreservoirs can function as a single-cell battery, and the distributionof multiple sets of adjacent, dissimilar reservoirs within the apparatuscan function as a field of single-cell batteries, which in the aggregateforms a multiple-cell battery distributed across a surface. Inembodiments utilizing an external power source the galvanic cell cancomprise electrodes connected to an external power source, for example abattery or other power source. In embodiments that areexternally-powered, the electrodes need not comprise dissimilarmaterials, as the external power source can define the anode andcathode. In certain externally powered embodiments, the power sourceneed not be physically connected to the device.

“Matrix” or “matrices” as used herein refer to a pattern or patterns,such as those formed by electrodes on a surface. Matrices can bedesigned to vary the electric field or electric microcurrent generated.For example, the strength and shape of the field or microcurrent can bealtered, or the matrices can be designed to produce an electric field(s)or current of a desired strength or shape.

“Reduction-oxidation reaction” or “redox reaction” as used herein refersto a reaction involving the transfer of one or more electrons from areducing agent to an oxidizing agent. The term “reducing agent” can bedefined in some embodiments as a reactant in a redox reaction, whichdonates electrons to a reduced species. A “reducing agent” is therebyoxidized in the reaction. The term “oxidizing agent” can be defined insome embodiments as a reactant in a redox reaction, which acceptselectrons from the oxidized species. An “oxidizing agent” is therebyreduced in the reaction. In various embodiments a redox reactionproduced between a first and second reservoir provides a current betweenthe dissimilar reservoirs. The redox reactions can occur spontaneouslywhen a conductive material is brought in proximity to first and seconddissimilar reservoirs such that the conductive material provides amedium for electrical communication and/or ionic communication betweenthe first and second dissimilar reservoirs. In other words, in anembodiment electrical currents can be produced between first and seconddissimilar reservoirs without the use of an external battery or otherpower source (e.g., a direct current (DC) such as a battery or analternating current (AC) power source such as a typical electricoutlet). Accordingly, in various embodiments a system is provided whichis “electrically self contained,” and yet the system can be activated toproduce electrical currents. The term “electrically self contained” canbe defined in some embodiments as being capable of producing electricity(e.g., producing currents) without an external battery or power source.The term “activated” can be defined in some embodiments to refer to theproduction of electric current through the application of a radio signalof a given frequency or through ultrasound or through electromagneticinduction. In other embodiments, a system can be provided which includesan external battery or power source. For example, an AC power source canbe of any wave form, such as a sine wave, a triangular wave, or a squarewave. AC power can also be of any frequency such as for example 50 Hz or60 HZ, or the like. AC power can also be of any voltage, such as forexample 120 volts, or 220 volts, or the like. In embodiments an AC powersource can be electronically modified, such as for example having thevoltage reduced, prior to use.

“Stretchable” as used herein refers to the ability of embodiments thatstretch without losing their structural integrity. That is, embodimentscan stretch to accommodate irregular wound surfaces or surfaces whereinone portion of the surface can move relative to another portion.

“Wound” as used herein includes abrasions, surgical incisions, cuts,punctures, tears, sores, ulcers, blisters, burns, amputations, bites,and any other breach or disruption of superficial tissue such as theskin, mucus membranes, epithelial linings, etc. Disruptions can includeinflamed areas, polyps, ulcers, etc. A scar is intended to includehypertrophic scars, keloids, or any healed wound tissue of the afflictedindividual. Superficial tissues include those tissues not normallyexposed in the absence of a wound or disruption, such as underlyingmuscle or connective tissue. A wound is not necessarily visible nor doesit necessarily involve rupture of superficial tissue, for example awound can comprise a bacterial infection. Wounds can include insect andanimal bites from both venomous and non-venomous insects and animals.

LLMC/LLEF Systems—Methods of Manufacture

A LLMC or LLEF system disclosed herein can comprise “anchor” regions or“arms” to affix the system securely. The anchor regions or arms cananchor the LLMC system, such as for example to areas around a jointwhere motion is minimal or limited. For example, a LLMC system can besecured to a wound proximal to a joint, and the anchor regions of thesystem can extend to areas of minimal stress or movement to securelyaffix the system. Further, the LLMC system can reduce stress on thewound site by “countering” the physical stress caused by movement. Forexample, the wound management system can be pre-stressed or stretchedprior to application such that it “pulls” or “holds” the wound perimetertogether.

A LLMC or LLEF system disclosed herein can comprise reinforcingsections. In embodiments the reinforcing sections can comprise sectionsthat span the length of the system. In embodiments a LLMC or LLEF systemcan comprise multiple reinforcing sections such as at least 1reinforcing section, at least 2 reinforcing sections, at least 3reinforcing sections, at least 4 reinforcing sections, at least 5reinforcing sections, at least 6 reinforcing sections, or the like.

In embodiments the LLMC or LLEF system can comprise additional materialsto aid in healing. These additional materials can comprise activationgels, rhPDGF (recombinant human platelet-derived growth factor)(REGRANEX®), Vibronectin:IGF complexes, CELLSPRAY (Clinical Cell CulturePty. Ltd., Australia), RECELL® (Clinical Cell Culture Pty. Ltd.,Australia), INTEGRA® dermal regeneration template (Integra LifeSciences, U.S.), BIOMEND® (Zimmer Dental Inc., U.S.), INFUSE® (MedtronicSofamor Danek Inc., U.S.), ALLODERM® (LifeCell Corp. U.S.), CYMETRA®(LifeCell Corp. U.S.), SEPRAPACK® (Genzyme Corporation, U.S.),SEPRAMESH® (Genzyme Corporation, U.S.), SKINTEMP® (Human BioSciencesInc., U.S.), COSMODERM® (Inamed Corporation, U.S.), COSMOPLAST® (InamedCorporation, U.S.), OP-1® (Stryker Corporation, U.S.), ISOLAGEN®(Fibrocell Technologies Inc., U.S.), CARTICEL® (Genzyme Corporation,U.S.), APLIGRAF® (Sandoz AG Corporation, Switzerland), DERMAGRAFT®(Smith & Nephew Wound Management Corporation, U.S.), TRANSCYTE® (ShireRegenerative Medicine Inc., U.S.), ORCEL® (Orcell LLPC Corporation,U.S.), EPICEL® (Genzyme Corporation, U.S.), and the like. In embodimentsthe additional materials can be, for example, TEGADERM® 91110 (3MCorporation, U.S.), MEPILEX® Normal Gel 0.9% Sodium chloride (MolnlyckeHealth Care AB, Sweden), HISPAGEL® (BASF Corporation, U.S.), LUBRIGEL®(Sheffield Laboratories Corporation, U.S.) or other compositions usefulfor maintaining a moist environment about a wound or for ease of removalof the LLMC or LLEF system. In certain embodiments additional materialsthat can be added to the LLMC or LLEF system can include for example,vesicular-based formulations such as hemoglobin vesicles. In certainembodiments liposome-based formulations can be used.

Embodiments can include devices in the form of a gel, such as, forexample, a one- or two-component gel that is mixed on use. Embodimentscan include devices in the form of a spray, for example, a one- ortwo-component spray that is mixed on use.

In embodiments, the LLMC or LLEF system can comprise instructions ordirections on how to place the system to maximize its performance.

Embodiments of the LLMC or LLEF system disclosed herein can compriseelectrodes or microcells. Each electrode or microcell can be or includea conductive metal. In embodiments, the electrodes or microcells cancomprise any electrically-conductive material, for example, anelectrically conductive hydrogel, metals, electrolytes, superconductors,semiconductors, plasmas, and nonmetallic conductors such as graphite andconductive polymers. Electrically conductive metals can include silver,copper, gold, aluminum, molybdenum, zinc, lithium, tungsten, brass,carbon, nickel, iron, palladium, platinum, tin, bronze, carbon steel,lead, titanium, stainless steel, mercury, Fe/Cr alloys, and the like.The electrode can be coated or plated with a different metal such asaluminum, gold, platinum or silver.

In certain embodiments reservoir or electrode geometry can comprisecircles, polygons, lines, zigzags, ovals, stars, or any suitable varietyof shapes. This provides the ability to design/customize surfaceelectric field shapes as well as depth of penetration.

Reservoir or dot sizes and concentrations can be of various sizes, asthese variations can allow for changes in the properties of the electricfield created by embodiments of the invention. Certain embodimentsprovide an electric field at about 1 Volt and then, under normal tissueloads with resistance of 100k to 300K ohms, produce a current in therange of 10 microamperes. The electric field strength can be determinedby calculating % the separation distance and applying it in the z-axisover the midpoint between the cell. This indicates the theoreticallocation of the highest strength field line.

In certain embodiments dissimilar metals can be used to create anelectric field with a desired voltage. In certain embodiments thepattern of reservoirs can control the watt density and shape of theelectric field.

In embodiments “ink” or “paint” can comprise any conductive solutionsuitable for forming an electrode on a surface, such as a conductivemetal solution. In embodiments “printing” or “painted” can comprise anymethod of applying a conductive material such as a conductive liquidmaterial to a material upon which a matrix is desired.

In embodiments printing devices can be used to produce LLMC or LLEFsystems disclosed herein. For example, inkjet or “3D” printers can beused to produce embodiments.

In certain embodiments the binders or inks used to produce LLMC or LLEFsystems disclosed herein can include, for example, poly cellulose inks,poly acrylic inks, poly urethane inks, silicone inks, and the like. Inembodiments the type of ink used can determine the release rate ofelectrons from the reservoirs. In embodiments various materials can beadded to the ink or binder such as, for example, conductive or resistivematerials can be added to alter the shape or strength of the electricfield. Other materials, such as silicon, can be added to enhance scarreduction. Such materials can also be added to the spaces betweenreservoirs.

Certain embodiments can utilize a power source to create the electriccurrent, such as a battery or a microbattery. The power source can beany energy source capable of generating a current in the LLMC system andcan include, for example, AC power, DC power, radio frequencies (RF)such as pulsed RF, induction, ultrasound, and the like.

Dissimilar metals used to make a LLMC or LLEF system disclosed hereincan be silver and zinc, and the electrolytic solution can include sodiumchloride in water. In certain embodiments the electrodes are appliedonto a non-conductive surface to create a pattern, most preferably anarray or multi-array of voltaic cells that do not spontaneously reactuntil they contact an electrolytic solution, for example wound fluid.Sections of this description use the terms “printing” with “ink,” but itis understood that the patterns may instead be “painted” with “paints.”The use of any suitable means for applying a conductive material iscontemplated. In embodiments “ink” or “paint” can comprise any solutionsuitable for forming an electrode on a surface such as a conductivematerial including a conductive metal solution. In embodiments“printing” or “painted” can comprise any method of applying a solutionto a material upon which a matrix is desired. It is also assumed that acompetent practitioner knows how to properly apply and cure thesolutions without any assistance, other than perhaps instructions thatshould be included with the selected binder that is used to make themixtures that will be used in the printing process.

A preferred material to use in combination with silver to create thevoltaic cells or reservoirs of disclosed embodiments is zinc. Zinc hasbeen well-described for its uses in prevention of infection in suchtopical antibacterial agents as Bacitracin zinc, a zinc salt ofBacitracin. Zinc is a divalent cation with antibacterial properties ofits own in addition to possessing the added benefit of being a cofactorto proteins of the metalloproteinase family of enzymes important to thephagocytic debridement and remodeling phases of wound healing. As acofactor zinc promotes and accelerates the functional activity of theseenzymes, resulting in better more efficient wound healing.

Turning to the figures, in FIG. 1, the dissimilar electrodes firstelectrode 6 and second electrode 10 are applied onto a desired primarysurface 2 of an article 4. In one embodiment primary surface is asurface of a LLMC or LLEF system that comes into direct contact with anarea to be treated such as skin surface or a wound. In alternateembodiments primary surface 2 is one which is desired to beantimicrobial, such as a medical instrument, implant, surgical gown,gloves, socks, table, door knob, or other surface that will contact anelectrolytic solution including sweat, so that at least part of thepattern of voltaic cells will spontaneously react and kill bacteria orother microbes.

In various embodiments the difference of the standard potentials of theelectrodes or dots or reservoirs can be in a range from 0.05 V toapproximately 5.0 V. For example, the standard potential can be 0.05 V,0.06 V, 0.07 V, 0.08 V, 0.09 V, 0.1 V, 0.2 V, or 0.3 V, 0.4 V, 0.5 V,0.6 V, 0.7 V, 0.8 V, 0.9 V, 1.0 V, 1.1 V, 1.2 V, 1.3 V, 1.4 V, 1.5 V,1.6 V, 1.7 V, 1.8 V, 1.9 V, 2.0 V, 2.1 V, 2.2 V, 2.3 V, 2.4 V, 2.5 V,2.6 V, 2.7 V, 2.8 V, 2.9 V, 3.0 V, 3.1 V, 3.2 V, 3.3 V, 3.4 V, 3.5 V,3.6 V, 3.7 V, 3.8 V, 3.9 V, 4.0 V, 4.1 V, 4.2 V, 4.3 V, 4.4 V, 4.5 V,4.6 V, 4.7 V, 4.8 V, 4.9 V, 5.0 V, 5.1 V, 5.2 V, 5.3 V, 5.4 V, 5.5 V,5.6 V, 5.7 V, 5.8 V, 5.9 V, 6.0 V, or the like.

In a particular embodiment, the difference of the standard potentials ofthe electrodes or dots or reservoirs can be at least 0.05 V, at least0.06 V, at least 0.07 V, at least 0.08 V, at least 0.09 V, at least 0.1V, at least 0.2 V, at least 0.3 V, at least 0.4 V, at least 0.5 V, atleast 0.6 V, at least 0.7 V, at least 0.8 V, at least 0.9 V, at least1.0 V, at least 1.1 V, at least 1.2 V, at least 1.3 V, at least 1.4 V,at least 1.5 V, at least 1.6 V, at least 1.7 V, at least 1.8 V, at least1.9 V, at least 2.0 V, at least 2.1 V, at least 2.2 V, at least 2.3 V,at least 2.4 V, at least 2.5 V, at least 2.6 V, at least 2.7 V, at least2.8 V, at least 2.9 V, at least 3.0 V, at least 3.1 V, at least 3.2 V,at least 3.3 V, at least 3.4 V, at least 3.5 V, at least 3.6 V, at least3.7 V, at least 3.8 V, at least 3.9 V, at least 4.0 V, at least 4.1 V,at least 4.2 V, at least 4.3 V, at least 4.4 V, at least 4.5 V, at least4.6 V, at least 4.7 V, at least 4.8 V, at least 4.9 V, at least 5.0 V,at least 5.1 V, at least 5.2 V, at least 5.3 V, at least 5.4 V, at least5.5 V, at least 5.6 V, at least 5.7 V, at least 5.8 V, at least 5.9 V,at least 6.0 V, or the like.

In a particular embodiment, the difference of the standard potentials ofthe electrodes or dots or reservoirs can be not more than 0.05 V, or notmore than 0.06 V, not more than 0.07 V, not more than 0.08 V, not morethan 0.09 V, not more than 0.1 V, not more than 0.2 V, not more than 0.3V, not more than 0.4 V, not more than 0.5 V, not more than 0.6 V, notmore than 0.7 V, not more than 0.8 V, not more than 0.9 V, not more than1.0 V, not more than 1.1 V, not more than 1.2 V, not more than 1.3 V,not more than 1.4 V, not more than 1.5 V, not more than 1.6 V, not morethan 1.7 V, not more than 1.8 V, not more than 1.9 V, not more than 2.0V, not more than 2.1 V, not more than 2.2 V, not more than 2.3 V, notmore than 2.4 V, not more than 2.5 V, not more than 2.6 V, not more than2.7 V, not more than 2.8 V, not more than 2.9 V, not more than 3.0 V,not more than 3.1 V, not more than 3.2 V, not more than 3.3 V, not morethan 3.4 V, not more than 3.5 V, not more than 3.6 V, not more than 3.7V, not more than 3.8 V, not more than 3.9 V, not more than 4.0 V, notmore than 4.1 V, not more than 4.2 V, not more than 4.3 V, not more than4.4 V, not more than 4.5 V, not more than 4.6 V, not more than 4.7 V,not more than 4.8 V, not more than 4.9 V, not more than 5.0 V, not morethan 5.1 V, not more than 5.2 V, not more than 5.3 V, not more than 5.4V, not more than 5.5 V, not more than 5.6 V, not more than 5.7 V, notmore than 5.8 V, not more than 5.9 V, not more than 6.0 V, or the like.

In embodiments, LLMC systems can produce a low level micro-current ofbetween for example about 1 and about 200 micro-amperes, between about10 and about 190 micro-amperes, between about 20 and about 180micro-amperes, between about 30 and about 170 micro-amperes, betweenabout 40 and about 160 micro-amperes, between about 50 and about 150micro-amperes, between about 60 and about 140 micro-amperes, betweenabout 70 and about 130 micro-amperes, between about 80 and about 120micro-amperes, between about 90 and about 100 micro-amperes, or thelike.

In embodiments, LLMC systems can produce a low level micro-current ofbetween for example about 1 and about 400 micro-amperes, between about20 and about 380 micro-amperes, between about 400 and about 360micro-amperes, between about 60 and about 340 micro-amperes, betweenabout 80 and about 320 micro-amperes, between about 100 and about 3000micro-amperes, between about 120 and about 280 micro-amperes, betweenabout 140 and about 260 micro-amperes, between about 160 and about 240micro-amperes, between about 180 and about 220 micro-amperes, or thelike.

In embodiments, LLMC systems of the invention can produce a low levelmicro-current about 10 micro-amperes, about 20 micro-amperes, about 30micro-amperes, about 40 micro-amperes, about 50 micro-amperes, about 60micro-amperes, about 70 micro-amperes, about 80 micro-amperes, about 90micro-amperes, about 100 micro-amperes, about 110 micro-amperes, about120 micro-amperes, about 130 micro-amperes, about 140 micro-amperes,about 150 micro-amperes, about 160 micro-amperes, about 170micro-amperes, about 180 micro-amperes, about 190 micro-amperes, about200 micro-amperes, about 210 micro-amperes, about 220 micro-amperes,about 240 micro-amperes, about 260 micro-amperes, about 280micro-amperes, about 300 micro-amperes, about 320 micro-amperes, about340 micro-amperes, about 360 micro-amperes, about 380 micro-amperes,about 400 micro-amperes, or the like.

In embodiments, LLMC systems can produce a low level micro-current ofnot more than 10 micro-amperes, or not more than 20 micro-amperes, notmore than 30 micro-amperes, not more than 40 micro-amperes, not morethan 50 micro-amperes, not more than 60 micro-amperes, not more than 70micro-amperes, not more than 80 micro-amperes, not more than 90micro-amperes, not more than 100 micro-amperes, not more than 110micro-amperes, not more than 120 micro-amperes, not more than 130micro-amperes, not more than 140 micro-amperes, not more than 150micro-amperes, not more than 160 micro-amperes, not more than 170micro-amperes, not more than 180 micro-amperes, not more than 190micro-amperes, not more than 200 micro-amperes, not more than 210micro-amperes, not more than 220 micro-amperes, not more than 230micro-amperes, not more than 240 micro-amperes, not more than 250micro-amperes, not more than 260 micro-amperes, not more than 270micro-amperes, not more than 280 micro-amperes, not more than 290micro-amperes, not more than 300 micro-amperes, not more than 310micro-amperes, not more than 320 micro-amperes, not more than 340micro-amperes, not more than 360 micro-amperes, not more than 380micro-amperes, not more than 400 micro-amperes, not more than 420micro-amperes, not more than 440 micro-amperes, not more than 460micro-amperes, not more than 480 micro-amperes, or the like.

In embodiments, LLMC systems of the invention can produce a low levelmicro-current of not less than 10 micro-amperes, not less than 20micro-amperes, not less than 30 micro-amperes, not less than 40micro-amperes, not less than 50 micro-amperes, not less than 60micro-amperes, not less than 70 micro-amperes, not less than 80micro-amperes, not less than 90 micro-amperes, not less than 100micro-amperes, not less than 110 micro-amperes, not less than 120micro-amperes, not less than 130 micro-amperes, not less than 140micro-amperes, not less than 150 micro-amperes, not less than 160micro-amperes, not less than 170 micro-amperes, not less than 180micro-amperes, not less than 190 micro-amperes, not less than 200micro-amperes, not less than 210 micro-amperes, not less than 220micro-amperes, not less than 230 micro-amperes, not less than 240micro-amperes, not less than 250 micro-amperes, not less than 260micro-amperes, not less than 270 micro-amperes, not less than 280micro-amperes, not less than 290 micro-amperes, not less than 300micro-amperes, not less than 310 micro-amperes, not less than 320micro-amperes, not less than 330 micro-amperes, not less than 340micro-amperes, not less than 350 micro-amperes, not less than 360micro-amperes, not less than 370 micro-amperes, not less than 380micro-amperes, not less than 390 micro-amperes, not less than 400micro-amperes, or the like.

The applied electrodes or reservoirs or dots can adhere or bond to theprimary surface 2 because a biocompatible binder is mixed, inembodiments into separate mixtures, with each of the dissimilar metalsthat will create the pattern of voltaic cells, in embodiments. Most inksare simply a carrier, and a binder mixed with pigment. Similarly,conductive metal solutions can be a binder mixed with a conductiveelement. The resulting conductive metal solutions can be used with anapplication method such as screen printing to apply the electrodes tothe primary surface in predetermined patterns. Once the conductive metalsolutions dry and/or cure, the patterns of spaced electrodes cansubstantially maintain their relative position, even on a flexiblematerial such as that used for a LLMC or LLEF system. To make a limitednumber of the systems of an embodiment disclosed herein, the conductivemetal solutions can be hand applied onto a common adhesive bandage sothat there is an array of alternating electrodes that are spaced about amillimeter apart on the primary surface of the bandage. The solutionshould be allowed to dry before being applied to a surface so that theconductive materials do not mix, which would destroy the array and causedirect reactions that will release the elements, but fail to simulatethe current of injury. However, the wound management system would stillexhibit an antimicrobial effect even if the materials were mixed.Furthermore, though silver alone will demonstrate antimicrobial effects,embodiments of the invention show antimicrobial activity greater thanthat of silver alone.

In certain embodiments that utilize a poly-cellulose binder, the binderitself can have an beneficial effect such as reducing the localconcentration of matrix metallo-proteases through an iontophoreticprocess that drives the cellulose into the surrounding tissue. Thisprocess can be used to electronically drive other components such asdrugs into the surrounding tissue.

The binder can include any biocompatible liquid material that can bemixed with a conductive element (preferably metallic crystals of silveror zinc) to create a conductive solution which can be applied as a thincoating to a surface. One suitable binder is a solvent reduciblepolymer, such as the polyacrylic non-toxic silk-screen ink manufacturedby COLORCON® Inc., a division of Berwind Pharmaceutical Services, Inc.(see COLORCON® NO-TOX® product line, part number NT28). In an embodimentthe binder is mixed with high purity (at least 99.999%) metallic silvercrystals to make the silver conductive solution. The silver crystals,which can be made by grinding silver into a powder, are preferablysmaller than 100 microns in size or about as fine as flour. In anembodiment, the size of the crystals is about 325 mesh, which istypically about 40 microns in size or a little smaller. The binder isseparately mixed with high purity (at least 99.99%, in an embodiment)metallic zinc powder which has also preferably been sifted throughstandard 325 mesh screen, to make the zinc conductive solution. Forbetter quality control and more consistent results, most of the crystalsused should be larger than 325 mesh and smaller than 200 mesh. Forexample the crystals used should be between 200 mesh and 325 mesh, orbetween 210 mesh and 310 mesh, between 220 mesh and 300 mesh, between230 mesh and 290 mesh, between 240 mesh and 280 mesh, between 250 meshand 270 mesh, between 255 mesh and 265 mesh, or the like.

Other powders of metal can be used to make other conductive metalsolutions in the same way as described in other embodiments.

The size of the metal crystals, the availability of the surface to theconductive fluid and the ratio of metal to binder affects the releaserate of the metal from the mixture. When COLORCON® polyacrylic ink isused as the binder, about 10 to 40 percent of the mixture should bemetal for a longer term bandage (for example, one that stays on forabout 10 days). For example, for a longer term LLMC or LLEF system thepercent of the mixture that should be metal can be 8 percent, or 10percent, 12 percent, 14 percent, 16 percent, 18 percent, 20 percent, 22percent, 24 percent, 26 percent, 28 percent, 30 percent, 32 percent, 34percent, 36 percent, 38 percent, 40 percent, 42 percent, 44 percent, 46percent, 48 percent, 50 percent, or the like.

If the same binder is used, but the percentage of the mixture that ismetal is increased to 60 percent or higher, then the release rate willbe much faster and a typical system will only be effective for a fewdays. For example, for a shorter term bandage, the percent of themixture that should be metal can be 40 percent, or 42 percent, 44percent, 46 percent, 48 percent, 50 percent, 52 percent, 54 percent, 56percent, 58 percent, 60 percent, 62 percent, 64 percent, 66 percent, 68percent, 70 percent, 72 percent, 74 percent, 76 percent, 78 percent, 80percent, 82 percent, 84 percent, 86 percent, 88 percent, 90 percent, orthe like.

It should be noted that polyacrylic ink can crack if applied as a verythin coat, which exposes more metal crystals which will spontaneouslyreact. For LLMC or LLEF systems comprising an article of clothing it maybe desired to decrease the percentage of metal down to 5 percent orless, or to use a binder that causes the crystals to be more deeplyembedded, so that the primary surface will be antimicrobial for a verylong period of time and will not wear prematurely. Other binders candissolve or otherwise break down faster or slower than a polyacrylicink, so adjustments can be made to achieve the desired rate ofspontaneous reactions from the voltaic cells.

To maximize the number of voltaic cells, in various embodiments, apattern of alternating silver masses or electrodes or reservoirs andzinc masses or electrodes or reservoirs can create an array ofelectrical currents across the primary surface. A basic pattern, shownin FIG. 1, has each mass of silver equally spaced from four masses ofzinc, and has each mass of zinc equally spaced from four masses ofsilver, according to an embodiment. The first electrode 6 is separatedfrom the second electrode 10 by a spacing 8. The designs of firstelectrode 6 and second electrode 10 are simply round dots, and in anembodiment, are repeated. Numerous repetitions 12 of the designs resultin a pattern. For a wound management system or dressing, each silverdesign preferably has about twice as much mass as each zinc design, inan embodiment. For the pattern in FIG. 1, the silver designs are mostpreferably about a millimeter from each of the closest four zincdesigns, and vice-versa. The resulting pattern of dissimilar metalmasses defines an array of voltaic cells when introduced to anelectrolytic solution. Further disclosure relating to methods ofproducing micro-arrays can be found in U.S. Pat. No. 7,813,806 entitledCURRENT PRODUCING SURFACE FOR TREATING BIOLOGIC TISSUE issued Oct. 12,2010, which is incorporated by reference in its entirety.

A dot pattern of masses like the alternating round dots of FIG. 1 can bepreferred when applying conductive material onto a flexible material,such as those used for a wound dressing, because the dots won'tsignificantly affect the flexibility of the material. The pattern ofFIG. 1 is well suited for general use. To maximize the density ofelectrical current over a primary surface the pattern of FIG. 2 can beused. The first electrode 6 in FIG. 2 is a large hexagonally shaped dot,and the second electrode 10 is a pair of smaller hexagonally shaped dotsthat are spaced from each other. The spacing 8 that is between the firstelectrode 6 and the second electrode 10 maintains a relativelyconsistent distance between adjacent sides of the designs. Numerousrepetitions 12 of the designs result in a pattern 14 that can bedescribed as at least one of the first design being surrounded by sixhexagonally shaped dots of the second design. The pattern of FIG. 2 iswell suited for abrasions and burns, as well as for insect bites,including those that can transfer bacteria or microbes or otherorganisms from the insect. There are of course other patterns that couldbe printed to achieve similar results.

FIGS. 3 and 4 show how the pattern of FIG. 2 can be used to make anadhesive bandage. The pattern shown in detail in FIG. 2 is applied tothe primary surface 2 of a wound dressing material. The back 20 of theprinted dressing material is fixed to an absorbent wound dressing layer22 such as cotton. The absorbent dressing layer is adhesively fixed toan elastic adhesive layer 16 such that there is at least one overlappingpiece or anchor 18 of the elastic adhesive layer that can be used tosecure the wound management system over a wound.

FIG. 5 shows an additional feature, which can be added between designs,that will start the flow of current in a poor electrolytic solution. Afine line 24 is printed using one of the conductive metal solutionsalong a current path of each voltaic cell. The fine line will initiallyhave a direct reaction but will be depleted until the distance betweenthe electrodes increases to where maximum voltage is realized. Theinitial current produced is intended to help control edema so that theLLMC system will be effective. If the electrolytic solution is highlyconductive when the system is initially applied the fine line can bequickly depleted and the wound dressing will function as though the fineline had never existed.

FIGS. 6 and 7 show alternative patterns that use at least one linedesign. The first electrode 6 of FIG. 6 is a round dot similar to thefirst design used in FIG. 1. The second electrode 10 of FIG. 6 is aline. When the designs are repeated, they define a pattern of parallellines that are separated by numerous spaced dots. FIG. 7 uses only linedesigns. The pattern of FIG. 7 is well suited for cuts, especially whenthe lines are perpendicular to a cut. The first electrode 6 can bethicker or wider than the second electrode 10 if the oxidation-reductionreaction requires more metal from the first conductive element (mixedinto the first design's conductive metal solution) than the secondconductive element (mixed into the second design's conductive metalsolution). The lines can be dashed. Another pattern can be silver gridlines that have zinc masses in the center of each of the cells of thegrid. The pattern can be letters printed from alternating conductivematerials so that a message can be printed onto the primarysurface-perhaps a brand name or identifying information such as patientblood type.

Because the spontaneous oxidation-reduction reaction of silver and zincuses a ratio of approximately two silver to one zinc, the silver designcan contain about twice as much mass as the zinc design in anembodiment. At a spacing of about 1 mm between the closest dissimilarmetals (closest edge to closest edge) each voltaic cell that is in woundfluid can create approximately 1 volt of potential that will penetratesubstantially through the dermis and epidermis. Closer spacing of thedots can decrease the resistance, providing less potential, and thecurrent will not penetrate as deeply. If the spacing falls below aboutone tenth of a millimeter a benefit of the spontaneous reaction is thatwhich is also present with a direct reaction; silver is electricallydriven into the wound, but the current of injury may not besubstantially simulated. Therefore, spacing between the closestconductive materials can be 0.1 mm, or 0.2 mm, 0.3 mm, 0.4 mm, 0.5 mm,0.6 mm, 0.7 mm, 0.8 mm, 0.9 mm, 1 mm, 1.1 mm, 1.2 mm, 1.3 mm, 1.4 mm,1.5 mm, 1.6 mm, 1.7 mm, 1.8 mm, 1.9 mm, 2 mm, 2.1 mm, 2.2 mm, 2.3 mm,2.4 mm, 2.5 mm, 2.6 mm, 2.7 mm, 2.8 mm, 2.9 mm, 3 mm, 3.1 mm, 3.2 mm,3.3 mm, 3.4 mm, 3.5 mm, 3.6 mm, 3.7 mm, 3.8 mm, 3.9 mm, 4 mm, 4.1 mm,4.2 mm, 4.3 mm, 4.4 mm, 4.5 mm, 4.6 mm, 4.7 mm, 4.8 mm, 4.9 mm, 5 mm,5.1 mm, 5.2 mm, 5.3 mm, 5.4 mm, 5.5 mm, 5.6 mm, 5.7 mm, 5.8 mm, 5.9 mm,6 mm, or the like.

In certain embodiments the spacing between the closest conductivematerials can be not more than 0.1 mm, or not more than 0.2 mm, not morethan 0.3 mm, not more than 0.4 mm, not more than 0.5 mm, not more than0.6 mm, not more than 0.7 mm, not more than 0.8 mm, not more than 0.9mm, not more than 1 mm, not more than 1.1 mm, not more than 1.2 mm, notmore than 1.3 mm, not more than 1.4 mm, not more than 1.5 mm, not morethan 1.6 mm, not more than 1.7 mm, not more than 1.8 mm, not more than1.9 mm, not more than 2 mm, not more than 2.1 mm, not more than 2.2 mm,not more than 2.3 mm, not more than 2.4 mm, not more than 2.5 mm, notmore than 2.6 mm, not more than 2.7 mm, not more than 2.8 mm, not morethan 2.9 mm, not more than 3 mm, not more than 3.1 mm, not more than 3.2mm, not more than 3.3 mm, not more than 3.4 mm, not more than 3.5 mm,not more than 3.6 mm, not more than 3.7 mm, not more than 3.8 mm, notmore than 3.9 mm, not more than 4 mm, not more than 4.1 mm, not morethan 4.2 mm, not more than 4.3 mm, not more than 4.4 mm, not more than4.5 mm, not more than 4.6 mm, not more than 4.7 mm, not more than 4.8mm, not more than 4.9 mm, not more than 5 mm, not more than 5.1 mm, notmore than 5.2 mm, not more than 5.3 mm, not more than 5.4 mm, not morethan 5.5 mm, not more than 5.6 mm, not more than 5.7 mm, not more than5.8 mm, not more than 5.9 mm, not more than 6 mm, or the like.

In certain embodiments spacing between the closest conductive materialscan be not less than 0.1 mm, or not less than 0.2 mm, not less than 0.3mm, not less than 0.4 mm, not less than 0.5 mm, not less than 0.6 mm,not less than 0.7 mm, not less than 0.8 mm, not less than 0.9 mm, notless than 1 mm, not less than 1.1 mm, not less than 1.2 mm, not lessthan 1.3 mm, not less than 1.4 mm, not less than 1.5 mm, not less than1.6 mm, not less than 1.7 mm, not less than 1.8 mm, not less than 1.9mm, not less than 2 mm, not less than 2.1 mm, not less than 2.2 mm, notless than 2.3 mm, not less than 2.4 mm, not less than 2.5 mm, not lessthan 2.6 mm, not less than 2.7 mm, not less than 2.8 mm, not less than2.9 mm, not less than 3 mm, not less than 3.1 mm, not less than 3.2 mm,not less than 3.3 mm, not less than 3.4 mm, not less than 3.5 mm, notless than 3.6 mm, not less than 3.7 mm, not less than 3.8 mm, not lessthan 3.9 mm, not less than 4 mm, not less than 4.1 mm, not less than 4.2mm, not less than 4.3 mm, not less than 4.4 mm, not less than 4.5 mm,not less than 4.6 mm, not less than 4.7 mm, not less than 4.8 mm, notless than 4.9 mm, not less than 5 mm, not less than 5.1 mm, not lessthan 5.2 mm, not less than 5.3 mm, not less than 5.4 mm, not less than5.5 mm, not less than 5.6 mm, not less than 5.7 mm, not less than 5.8mm, not less than 5.9 mm, not less than 6 mm, or the like.

Disclosures of the present specification include LLMC or LLEF systemscomprising a primary surface of a pliable material wherein the pliablematerial is adapted to be applied to an area of tissue; a firstelectrode design formed from a first conductive liquid that includes amixture of a polymer and a first element, the first conductive liquidbeing applied into a position of contact with the primary surface, thefirst element including a metal species, and the first electrode designincluding at least one dot or reservoir, wherein selective ones of theat least one dot or reservoir have approximately a 1.5 mm+/−1 mm meandiameter; a second electrode design formed from a second conductiveliquid that includes a mixture of a polymer and a second element, thesecond element including a different metal species than the firstelement, the second conductive liquid being printed into a position ofcontact with the primary surface, and the second electrode designincluding at least one other dot or reservoir, wherein selective ones ofthe at least one other dot or reservoir have approximately a 2.5 mm+/−2mm mean diameter; a spacing on the primary surface that is between thefirst electrode design and the second electrode design such that thefirst electrode design does not physically contact the second electrodedesign, wherein the spacing is approximately 1.5 mm+/−1 mm, and at leastone repetition of the first electrode design and the second electrodedesign, the at least one repetition of the first electrode design beingsubstantially adjacent the second electrode design, wherein the at leastone repetition of the first electrode design and the second electrodedesign, in conjunction with the spacing between the first electrodedesign and the second electrode design, defines at least one pattern ofat least one voltaic cell for spontaneously generating at least oneelectrical current when introduced to an electrolytic solution.Therefore, electrodes, dots or reservoirs can have a mean diameter of0.2 mm, or 0.3 mm, 0.4 mm, 0.5 mm, 0.6 mm, 0.7 mm, 0.8 mm, 0.9 mm, 1.0mm, 1.1 mm, 1.2 mm, 1.3 mm, 1.4 mm, 1.5 mm, 1.6 mm, 1.7 mm, 1.8 mm, 1.9mm, 2.0 mm, 2.1 mm, 2.2 mm, 2.3 mm, 2.4 mm, 2.5 mm, 2.6 mm, 2.7 mm, 2.8mm, 2.9 mm, 3.0 mm, 3.1 mm, 3.2 mm, 3.3 mm, 3.4 mm, 3.5 mm, 3.6 mm, 3.7mm, 3.8 mm, 3.9 mm, 4.0 mm, 4.1 mm, 4.2 mm, 4.3 mm, 4.4 mm, 4.5 mm, 4.6mm, 4.7 mm, 4.8 mm, 4.9 mm, 5.0 mm, or the like.

In further embodiments, electrodes, dots or reservoirs can have a meandiameter of not less than 0.2 mm, or not less than 0.3 mm, not less than0.4 mm, not less than 0.5 mm, not less than 0.6 mm, not less than 0.7mm, not less than 0.8 mm, not less than 0.9 mm, not less than 1.0 mm,not less than 1.1 mm, not less than 1.2 mm, not less than 1.3 mm, notless than 1.4 mm, not less than 1.5 mm, not less than 1.6 mm, not lessthan 1.7 mm, not less than 1.8 mm, not less than 1.9 mm, not less than2.0 mm, not less than 2.1 mm, not less than 2.2 mm, not less than 2.3mm, not less than 2.4 mm, not less than 2.5 mm, not less than 2.6 mm,not less than 2.7 mm, not less than 2.8 mm, not less than 2.9 mm, notless than 3.0 mm, not less than 3.1 mm, not less than 3.2 mm, not lessthan 3.3 mm, not less than 3.4 mm, not less than 3.5 mm, not less than3.6 mm, not less than 3.7 mm, not less than 3.8 mm, not less than 3.9mm, not less than 4.0 mm, not less than 4.1 mm, not less than 4.2 mm,not less than 4.3 mm, not less than 4.4 mm, not less than 4.5 mm, notless than 4.6 mm, not less than 4.7 mm, not less than 4.8 mm, not lessthan 4.9 mm, not less than 5.0 mm, or the like.

In further embodiments, electrodes, dots or reservoirs can have a meandiameter of not more than 0.2 mm, or not more than 0.3 mm, not more than0.4 mm, not more than 0.5 mm, not more than 0.6 mm, not more than 0.7mm, not more than 0.8 mm, not more than 0.9 mm, not more than 1.0 mm,not more than 1.1 mm, not more than 1.2 mm, not more than 1.3 mm, notmore than 1.4 mm, not more than 1.5 mm, not more than 1.6 mm, not morethan 1.7 mm, not more than 1.8 mm, not more than 1.9 mm, not more than2.0 mm, not more than 2.1 mm, not more than 2.2 mm, not more than 2.3mm, not more than 2.4 mm, not more than 2.5 mm, not more than 2.6 mm,not more than 2.7 mm, not more than 2.8 mm, not more than 2.9 mm, notmore than 3.0 mm, not more than 3.1 mm, not more than 3.2 mm, not morethan 3.3 mm, not more than 3.4 mm, not more than 3.5 mm, not more than3.6 mm, not more than 3.7 mm, not more than 3.8 mm, not more than 3.9mm, not more than 4.0 mm, not more than 4.1 mm, not more than 4.2 mm,not more than 4.3 mm, not more than 4.4 mm, not more than 4.5 mm, notmore than 4.6 mm, not more than 4.7 mm, not more than 4.8 mm, not morethan 4.9 mm, not more than 5.0 mm, or the like.

The material concentrations or quantities within and/or the relativesizes (e.g., dimensions or surface area) of the first and secondreservoirs can be selected deliberately to achieve variouscharacteristics of the systems' behavior. For example, the quantities ofmaterial within a first and second reservoir can be selected to providean apparatus having an operational behavior that depletes atapproximately a desired rate and/or that “dies” after an approximateperiod of time after activation. In an embodiment the one or more firstreservoirs and the one or more second reservoirs are configured tosustain one or more currents for an approximate pre-determined period oftime, after activation. It is to be understood that the amount of timethat currents are sustained can depend on external conditions andfactors (e.g., the quantity and type of activation material), andcurrents can occur intermittently depending on the presence or absenceof activation material. Further disclosure relating to producingreservoirs that are configured to sustain one or more currents for anapproximate pre-determined period of time can be found in U.S. Pat. No.7,904,147 entitled SUBSTANTIALLY PLANAR ARTICLE AND METHODS OFMANUFACTURE issued Mar. 8, 2011, which is incorporated by referenceherein in its entirety.

In various embodiments the difference of the standard potentials of thefirst and second reservoirs can be in a range from 0.05 V toapproximately 5.0 V. For example, the standard potential can be 0.05 V,or 0.06 V, 0.07 V, 0.08 V, 0.09 V, 0.1 V, 0.2 V, 0.3 V, 0.4 V, 0.5 V,0.6 V, 0.7 V, 0.8 V, 0.9 V, 1.0 V, 1.1 V, 1.2 V, 1.3 V, 1.4 V, 1.5 V,1.6 V, 1.7 V, 1.8 V, 1.9 V, 2.0 V, 2.1 V, 2.2 V, 2.3 V, 2.4 V, 2.5 V,2.6 V, 2.7 V, 2.8 V, 2.9 V, 3.0 V, 3.1 V, 3.2 V, 3.3 V, 3.4 V, 3.5 V,3.6 V, 3.7 V, 3.8 V, 3.9 V, 4.0 V, 4.1 V, 4.2 V, 4.3 V, 4.4 V, 4.5 V,4.6 V, 4.7 V, 4.8 V, 4.9 V, 5.0 V, or the like.

In a particular embodiment the difference of the standard potentials ofthe first and second reservoirs can be at least 0.05 V, or at least 0.06V, at least 0.07 V, at least 0.08 V, at least 0.09 V, at least 0.1 V, atleast 0.2 V, at least 0.3 V, at least 0.4 V, at least 0.5 V, at least0.6 V, at least 0.7 V, at least 0.8 V, at least 0.9 V, at least 1.0 V,at least 1.1 V, at least 1.2 V, at least 1.3 V, at least 1.4 V, at least1.5 V, at least 1.6 V, at least 1.7 V, at least 1.8 V, at least 1.9 V,at least 2.0 V, at least 2.1 V, at least 2.2 V, at least 2.3 V, at least2.4 V, at least 2.5 V, at least 2.6 V, at least 2.7 V, at least 2.8 V,at least 2.9 V, at least 3.0 V, at least 3.1 V, at least 3.2 V, at least3.3 V, at least 3.4 V, at least 3.5 V, at least 3.6 V, at least 3.7 V,at least 3.8 V, at least 3.9 V, at least 4.0 V, at least 4.1 V, at least4.2 V, at least 4.3 V, at least 4.4 V, at least 4.5 V, at least 4.6 V,at least 4.7 V, at least 4.8 V, at least 4.9 V, at least 5.0 V, or thelike.

In a particular embodiment, the difference of the standard potentials ofthe first and second reservoirs can be not more than 0.05 V, or not morethan 0.06 V, not more than 0.07 V, not more than 0.08 V, not more than0.09 V, not more than 0.1 V, not more than 0.2 V, not more than 0.3 V,not more than 0.4 V, not more than 0.5 V, not more than 0.6 V, not morethan 0.7 V, not more than 0.8 V, not more than 0.9 V, not more than 1.0V, not more than 1.1 V, not more than 1.2 V, not more than 1.3 V, notmore than 1.4 V, not more than 1.5 V, not more than 1.6 V, not more than1.7 V, not more than 1.8 V, not more than 1.9 V, not more than 2.0 V,not more than 2.1 V, not more than 2.2 V, not more than 2.3 V, not morethan 2.4 V, not more than 2.5 V, not more than 2.6 V, not more than 2.7V, not more than 2.8 V, not more than 2.9 V, not more than 3.0 V, notmore than 3.1 V, not more than 3.2 V, not more than 3.3 V, not more than3.4 V, not more than 3.5 V, not more than 3.6 V, not more than 3.7 V,not more than 3.8 V, not more than 3.9 V, not more than 4.0 V, not morethan 4.1 V, not more than 4.2 V, not more than 4.3 V, not more than 4.4V, not more than 4.5 V, not more than 4.6 V, not more than 4.7 V, notmore than 4.8 V, not more than 4.9 V, not more than 5.0 V, or the like.In embodiments that include very small reservoirs (e.g., on thenanometer scale), the difference of the standard potentials can besubstantially less or more. The electrons that pass between the firstreservoir and the second reservoir can be generated as a result of thedifference of the standard potentials. Further disclosure relating tostandard potentials can be found in U.S. Pat. No. 8,224,439 entitledBATTERIES AND METHODS OF MANUFACTURE AND USE issued Jul. 17, 2012, whichis incorporated be reference herein in its entirety.

The voltage present at the site of treatment is typically in the rangeof millivolts but disclosed embodiments can introduce a much highervoltage, for example near 1 volt when using the 1 mm spacing ofdissimilar metals already described. The higher voltage is believed todrive the current deeper into the treatment area so that dermis andepidermis benefit from the simulated current of injury. In this way thecurrent not only can drive silver and zinc into the treatment, but thecurrent can also provide a stimulatory current so that the entiresurface area can heal simultaneously. In embodiments the current can,for example, kill microbes.

Embodiments disclosed herein relating to treatment of diseases orconditions or symptoms can also comprise selecting a patient or tissuein need of, or that could benefit by, treatment of that disease,condition, or symptom.

While various embodiments have been shown and described, it will berealized that alterations and modifications can be made thereto withoutdeparting from the scope of the following claims. For example it can bedesirable to use methods other than a common screen printing machine toapply the electrodes onto surfaces on medical instruments, garments,implants and the like so that they are antimicrobial. It is expectedthat other methods of applying the conductive material can besubstituted as appropriate. Also, there are numerous shapes, sizes andpatterns of voltaic cells that have not been described but it isexpected that this disclosure will enable those skilled in the art toincorporate their own designs which will then be applied to a surface tocreate voltaic cells which will become active when brought into contactwith an electrolytic solution.

Certain embodiments include LLMC or LLEF systems comprising dressings orbandages designed to be used on irregular, non-planar, or “stretching”surfaces such as joints. Embodiments disclosed herein can be used withnumerous joints of the body, including the jaw, the shoulder, the elbow,the wrist, the finger joints, the hip, the knee, the ankle, the toejoints, etc. Additional embodiments disclosed herein can be used inareas where tissue is prone to movement, for example the eyelid, theear, the lips, the nose, genitalia, etc.

Various apparatus embodiments which can be referred to as “medicalbatteries” are described herein. Further disclosure relating to thistechnology can be found in U.S. Pat. No. 7,672,719 entitled BATTERIESAND METHODS OF MANUFACTURE AND USE issued Mar. 2, 2010, which isincorporated herein by reference in its entirety.

Certain embodiments disclosed herein include a method of manufacturing asubstantially planar LLMC or LLEF system, the method comprising joiningwith a substrate multiple first reservoirs wherein selected ones of themultiple first reservoirs include a reducing agent, and wherein firstreservoir surfaces of selected ones of the multiple first reservoirs areproximate to a first substrate surface; and joining with the substratemultiple second reservoirs wherein selected ones of the multiple secondreservoirs include an oxidizing agent, and wherein second reservoirsurfaces of selected ones of the multiple second reservoirs areproximate to the first substrate surface, wherein joining the multiplefirst reservoirs and joining the multiple second reservoirs comprisesjoining using tattooing. In embodiments the substrate can comprisegauzes comprising dots or electrodes.

Further embodiments can include a method of manufacturing a LLMC or LLEFsystem, the method comprising joining with a substrate multiple firstreservoirs wherein selected ones of the multiple first reservoirsinclude a reducing agent, and wherein first reservoir surfaces ofselected ones of the multiple first reservoirs are proximate to a firstsubstrate surface; and joining with the substrate multiple secondreservoirs wherein selected ones of the multiple second reservoirsinclude an oxidizing agent, and wherein second reservoir surfaces ofselected ones of the multiple second reservoirs are proximate to thefirst substrate surface, wherein joining the multiple first reservoirsand joining the multiple second reservoirs comprises: combining themultiple first reservoirs, the multiple second reservoirs, and multipleparallel insulators to produce a pattern repeat arranged in a firstdirection across a plane, the pattern repeat including a sequence of afirst one of the parallel insulators, one of the multiple firstreservoirs, a second one of the parallel insulators, and one of themultiple second reservoirs; and weaving multiple transverse insulatorsthrough the first parallel insulator, the one first reservoir, thesecond parallel insulator, and the one second reservoir in a seconddirection across the plane to produce a woven apparatus.

LLMC/LLEF Systems—Methods of Use

Embodiments disclosed herein include LLMC and LLEF systems that canproduce an electrical stimulus and/or can electromotivate,electroconduct, electroinduct, electrotransport, and/or electrophoreseone or more therapeutic materials in areas of target tissue (e.g.,iontophoresis), and/or can cause one or more biologic or other materialsin proximity to, on or within target tissue to be affected (e.g.,attract, repel, kill, neutralize, or alter cellulargrowth/viability/mobility, etc.). Further disclosure relating tomaterials that can produce an electrical stimulus can be found in U.S.Pat. No. 7,662,176 entitled FOOTWEAR APPARATUS AND METHODS OFMANUFACTURE AND USE issued Feb. 16, 2010, which is incorporated hereinby reference in its entirety.

Treatment of Wounds

The wound healing process includes several phases, including aninflammatory phase and a proliferative phase. The proliferative phaseinvolves cell migration (such as by human keratinocytes) wherein cellsmigrate into the wound site and cell proliferation wherein the cellsreproduce. This phase also involves angiogenesis and the growth ofgranulation tissue. During cell migration, many epithelial cells havethe ability to detect electric fields and respond with directedmigration. Their response typically requires Ca²⁺ influx, the presenceof specific growth factors such as Integrin and intracellular kinaseactivity. Most types of cells migrate directionally in a small electricfield, a phenomenon called galvanotaxis or electrotaxis. Electric fieldsof strength equal to those detected at wound edges direct cell migrationand can override some other well-accepted coexistent guidance cues suchas contact inhibition. Aspects of the present specification disclose inpart a method of treating an individual with a wound. Treating a woundcan include covering the wound with a LLMC or LLEF system. Embodimentsdisclosed herein can promote wound healing by directing cell migrationduring the wound healing process.

In embodiments a wound can be an acute or chronic wound, a diabeticwound of the lower extremities, such as of the legs or feet, apost-radiation tissue injury, crush injuries or compartment syndrome andother acute traumatic ischemias, venous stasis or arterial-insufficiencyulcers, compromised grafts and flaps, infected wounds, pressure ulcers,necrotizing soft-tissue infections, burns, cancer-related wounds,osteomyelitis, surgical wounds, traumatic wounds, insect bites, and thelike. In an embodiment a wound can be a non-penetrating wound, such asthe result of blunt trauma or friction with other surfaces. Typicallythis type of wound does not break through the skin and may include anabrasion (scraping of the outer skin layer), a laceration (a tear-likewound), a contusion (swollen bruises due to accumulation of blood anddead cells under skin), or the like. In other embodiments a wound can bea penetrating wound. These result from trauma that breaks through thefull thickness of skin and include stab wounds (trauma from sharpobjects, such as knives), skin cuts, surgical wounds (intentional cutsin the skin to perform surgical procedures), shrapnel wounds (woundsresulting from exploding shells), or gunshot wounds (wounds resultingfrom firearms). In further embodiments a wound can be a thermal woundsuch as resulting from heat or cold, a chemical wound such as resultingfrom an acid or base, an electrical wound, or the like.

Chronic wounds often do not heal in normal stages, and the wounds canalso fail to heal in a timely fashion. LLMC and LLEF systems disclosedherein can promote the healing of chronic wounds by increasing cellmigration, cell proliferation, and/or cell signaling. Increasedmigration can be seen in various cell types, such as for examplekeratinocytes.

In embodiments, treating the wound can comprise applying a LLMC or LLEFsystem to the wound such that the system can stretch with movement ofthe wound and surrounding area. In certain embodiments, the system canbe stretched before application to the wound such that the woundmanagement system “pulls” the wound edges together.

In embodiments, methods for treating or dressing a wound comprises thestep of topically administering an additional material on the woundsurface or upon the matrix of biocompatible microcells. These additionalmaterials can comprise, for example, activation gels, rhPDGF(REGRANEX®), Vibronectin:IGF complexes, CELLSPRAY®, RECELL®, INTEGRA®dermal regeneration template, BIOMEND®, INFUSE®, ALLODERM®, CYMETRA®,SEPRAPACK®, SEPRAMESH®, SKINTEMP®, MEDFIL®, COSMODERM®, COSMOPLAST®,OP-1®, ISOLAGEN®, CARTICEL®, APLIGRAF®, DERMAGRAFT®, TRANSCYTE®, ORCEL®,EPICEL®, and the like. In embodiments the activation gel can be, forexample, TEGADERM® 91110 by 3M, MEPILEX® Normal Gel 0.9% Sodiumchloride, HISPAGEL®, LUBRIGEL®, or other compositions useful formaintaining a moist environment about the wound or useful for healing awound via another mechanism.

Aspects of the present specification provide, in part, methods ofreducing a symptom associated with a wound. In an aspect of thisembodiment the symptom reduced is edema, hyperemia, erythema, bruising,tenderness, stiffness, swollenness, fever, a chill, a breathing problem,fluid retention, a blood clot, a loss of appetite, an increased heartrate, a formation of granulomas, fibrinous, pus, or non-viscous serousfluid, a formation of an ulcer, or pain.

Treating a wound can refer to reducing the size of, or preventing anincrease in size of a wound. For example, treating can reduce the widthof a wound by, e.g., at least 20%, at least 25%, at least 30%, at least35%, at least 40%, at least 45%, at least 50%, at least 55%, at least60%, at least 65%, at least 70%, at least 75%, at least 80%, at least85%, at least 90% at least 95%, or at least 100%.

Treating a wound can refer to reducing the depth of, or preventing anincrease in depth of a wound. For example, treating can reduce the depthof a wound by, e.g., at least 20%, at least 25%, at least 30%, at least35%, at least 40%, at least 45%, at least 50%, at least 55%, at least60%, at least 65%, at least 70%, at least 75%, at least 80%, at least85%, at least 90% at least 95%, or at least 100%.

Treatment of Bites

Systems disclosed herein can be used to treat animal bites, for examplesnake bites. A LLMC or LLEF system can be applied to the bite(s) orbitten area, wherein the low level micro-current or electric field canneutralize the immune reaction to the bites or the venom, or neutralizethe antigens present in such bites and thus reduce pain and itching. Inembodiments the systems and devices disclosed herein can treat venomousbites by altering the function of venoms, such as, for example,protein-based venoms.

Systems disclosed herein can be used to treat insect bites, for examplemosquito bites. A LLMC or LLEF system can be applied to the bite(s) orbitten area, wherein the low level micro-current or electric field canneutralize the immune reaction to the bites or any venom and thus reducepain and itching.

Treatment of Microbial Infection

Embodiments of the disclosed LLMC and LLEF systems can providemicrobicidal activity. For example, embodiments disclosed herein canprevent, limit, or reduce formation of biofilms by interfering withbacterial signaling. Further embodiments can kill bacteria through anestablished biofilm.

Embodiments of the disclosed LLMC and LLEF systems can providemicrobicidal activity. For example, embodiments disclosed herein canprevent, limit, or reduce formation of biofilms by interfering withbacterial signaling. Further embodiments can kill bacteria through anestablished biofilm.

Aspects disclosed herein include systems, devices, and methods fortreating parasitic infections. For example, methods disclosed hereininclude treatments for ectoparasitic infections caused by, for example,Sarcoptes scabiei (causes scabies), Pediculus humanus capitis (causeshead lice), Phthirus pubis (causes pubic lice), Leishmania (causesleishmaniasis), and the like. Leishmaniasis is a highly focal diseasewith widely scattered foci. The parasite may survive for decades inasymptomatic infected people, who are of great importance for thetransmission since they can spread visceral leishmaniasis indirectlythrough the sandflies. The parasites can also be transmitted directlyfrom person to person through the sharing of infected needles which isoften the case with the Leishmania/HIV co-infection. Cutaneousleishmaniasis is the most common form, which causes a sore at the bitesite, which heals in a few months to a year, leaving anunpleasant-looking scar. Systems disclosed herein can be used to treatcutaneous leishmaniasis in the initial infection stage as well as thelatent stage or in the active disfiguring lesions resulting from theinfection.

Cellular Activation

Embodiments of the disclosed LLMC and LLEF systems can increase cellmigration by applying an electric current or electric field or both to atreatment area. For example, the systems can increase migration of humankeratinocytes. The systems can also be used to promotere-epithelialization for example in a wound.

Embodiments of the disclosed LLMC and LLEF systems can increase glucoseuptake in target tissues and cells, for example by applying a LLEFsystem disclosed herein to a treatment area where increased uptake ofglucose is desired. In embodiments glucose uptake can be increased toenergize mitochondria.

Embodiments of the disclosed LLMC and LLEF systems can increase cellsignaling in target tissues and cells, for example by applying a LLEFsystem disclosed herein to a treatment area where increased cellsignaling is desired.

Embodiments of the disclosed LLMC and LLEF systems can create hydrogenperoxide in target tissues and cells, for example by applying a LLEFsystem disclosed herein to a treatment area where hydrogen peroxideproduction is desired.

Treatment of Disease

Embodiments of the disclosed LLMC and LLEF systems can be used to treatdisease. For example, embodiments can be used to increase glucose uptakethus reducing serum glucose levels and treating diseases relating toincreased glucose levels, such as diabetes. Increasing cellular uptakeof glucose can also have a limiting effect on glucose level variations(excursions), thus treating both hyper- and hypoglycemia. Inembodiments, methods of treating glucose-related diseases can compriseapplying systems of the invention to a patient in need thereof. Forexample, LLEF or LLMC systems can be applied to a patient's skin, orapplied using a catheter, or applied using a pharmaceutical composition.A pharmaceutical composition disclosed herein can be administered to anindividual using a variety of routes. Routes of administration suitablefor use as disclosed herein include both local and systemicadministration. Local administration results in significantly moredelivery of a composition to a specific location as compared to theentire body of the individual, whereas, systemic administration resultsin delivery of a composition to essentially the entire body of theindividual.

Muscle Regeneration

Embodiments of the disclosed LLMC and LLEF systems can be used toregenerate muscle tissue. For example, embodiments can be used to directmacrophage migration to damaged or wounded muscle thus helping toregenerate the muscle.

EXAMPLES

The following non-limiting examples are provided for illustrativepurposes only in order to facilitate a more complete understanding ofrepresentative embodiments. These examples should not be construed tolimit any of the embodiments described in the present specificationincluding those pertaining to the methods of treating wounds.

Example 1 Cell Migration Assay

The in vitro scratch assay is an easy, low-cost and well-developedmethod to measure cell migration in vitro. The basic steps involvecreating a “scratch” in a cell monolayer, capturing images at thebeginning and at regular intervals during cell migration to close thescratch, and comparing the images to quantify the migration rate of thecells. Compared to other methods, the in vitro scratch assay isparticularly suitable for studies on the effects of cell-matrix andcell-cell interactions on cell migration, mimic cell migration duringwound healing in vivo and are compatible with imaging of live cellsduring migration to monitor intracellular events if desired. In additionto monitoring migration of homogenous cell populations, this method hasalso been adopted to measure migration of individual cells in theleading edge of the scratch. Not taking into account the time fortransfection of cells, in vitro scratch assay per se usually takes fromseveral hours to overnight.

Human keratinocytes were plated under plated under placebo or a LLMCsystem (labeled “PROCELLERA®”). Cells were also plated under silver-onlyor zinc-only dressings. After 24 hours, the scratch assay was performed.Cells plated under the PROCELLERA® device displayed increased migrationinto the “scratched” area as compared to any of the zinc, silver, orplacebo dressings. After 9 hours, the cells plated under the PROCELLERA®device had almost “closed” the scratch. This demonstrates the importanceof electrical activity to cell migration and infiltration.

In addition to the scratch test, genetic expression was tested.Increased insulin growth factor (IGF)-1R phosphorylation wasdemonstrated by the cells plated under the PROCELLERA® device ascompared to cells plated under insulin growth factor alone.

Integrin accumulation also affects cell migration. An increase inintegrin accumulation achieved with the LLMC system. Integrin isnecessary for cell migration, and is found on the leading edge ofmigrating cell.

Thus, the tested LLMC system enhanced cellular migration andIGF-1R/integrin involvement. This involvement demonstrates the effectthat the LLMC system had upon cell receptors involved with the woundhealing process.

Example 2 Zone of Inhibition Test

For cellular repair to be most efficient, available energy should not beshared with ubiquitous microbes. In this “zone of inhibition” test,placebo, a LLMC device (PROCELLERA®) and silver only were tested in anagar medium with a 24 hour growth of organisms. Bacterial growth waspresent over the placebo, a zone of inhibition over the PROCELLERA® anda minimal inhibition zone over the silver. Because the samples were“buried” in agar, the electricidal effect of the LLMC system could betested. This could mean the microbes were affected by the electricalfield or the silver ion transport through the agar was enhanced in thepresence of the electric field. Silver ion diffusion, the method used bysilver based antimicrobials, alone was not sufficient. The testdemonstrates the improved bactericidal effect of PROCELLERA® as comparedto silver alone.

Example 3 Wound Care Study

The medical histories of patients who received “standard-of-care”woundtreatment (“SOC”; n=20), or treatment with a LLMC device as disclosedherein (n=18), were reviewed. The wound care device used in the presentstudy consisted of a discrete matrix of silver and zinc dots. Asustained voltage of approximately 0.8 V was generated between the dots.The electric field generated at the device surface was measured to be0.2-1.0 V, 10-50 μA.

Wounds were assessed until closed or healed. The number of days to woundclosure and the rate of wound volume reduction were compared. Patientstreated with LLMC received one application of the device each week, ormore frequently in the presence of excessive wound exudate, inconjunction with appropriate wound care management. The LLMC was keptmoist by saturating with normal saline or conductive hydrogel.Adjunctive therapies (such as negative pressure wound therapy [NPWT],etc.) were administered with SOC or with the use of LLMC unlesscontraindicated. The SOC group received the standard of care appropriateto the wound, for example antimicrobial dressings, barrier creams,alginates, silver dressings, absorptive foam dressings, hydrogel,enzymatic debridement ointment, NPWT, etc. Etiology-specific care wasadministered on a case-by-case basis. Dressings were applied at weeklyintervals or more. The SOC and LLMC groups did not differ significantlyin gender, age, wound types or the length, width, and area of theirwounds.

Wound dimensions were recorded at the beginning of the treatment, aswell as interim and final patient visits. Wound dimensions, includinglength (L), width (W) and depth (D) were measured, with depth measuredat the deepest point. Wound closure progression was also documentedthrough digital photography. Determining the area of the wound wasperformed using the length and width measurements of the wound surfacearea.

Closure was defined as 100% epithelialization with visible effacement ofthe wound. Wounds were assessed 1 week post-closure to ensure continuedprogress toward healing during its maturation and remodeling phase.

Wound types included in this study were diverse in etiology anddimensions, thus the time to heal for wounds was distributed over a widerange (9-124 days for SOC, and 3-44 days for the LLMC group).Additionally, the patients often had multiple co-morbidities, includingdiabetes, renal disease, and hypertension. The average number of days towound closure was 36.25 (SD=28.89) for the SOC group and 19.78(SD=14.45) for the LLMC group, p=0.036. On average, the wounds in theLLMC treatment group attained closure 45.43% earlier than those in theSOC group.

Based on the volume calculated, some wounds improved persistently whileothers first increased in size before improving. The SOC and the LLMCgroups were compared to each other in terms of the number of instanceswhen the dimensions of the patient wounds increased (i.e., woundtreatment outcome degraded). In the SOC group, 10 wounds (50% for n=20)became larger during at least one measurement interval, whereas 3 wounds(16.7% for n=18) became larger in the LLMC group (p=0.018). Overall,wounds in both groups responded positively. Response to treatment wasobserved to be slower during the initial phase, but was observed toimprove as time progressed.

The LLMC wound treatment group demonstrated on average a 45.4% fasterclosure rate as compared to the SOC group. Wounds receiving SOC weremore likely to follow a “waxing-and-waning” progression in wound closurecompared to wounds in the LLMC treatment group.

Compared to localized SOC treatments for wounds, the LLMC (1) reduceswound closure time, (2) has a steeper wound closure trajectory, and (3)has a more robust wound healing trend with fewer incidence of increasedwound dimensions during the course of healing.

Example 4 LLMC Influence on Human Keratinocyte Migration

An LLMC -generated electrical field was mapped, leading to theobservation that LLMC generates hydrogen peroxide, known to drive redoxsignaling. LLMC-induced phosphorylation of redox-sensitive IGF-1R wasdirectly implicated in cell migration. The LLMC also increasedkeratinocyte mitochondrial membrane potential.

The LLMC was made of polyester printed with dissimilar elemental metals.It comprises alternating circular regions of silver and zinc dots, alongwith a proprietary, biocompatible binder added to lock the electrodes tothe surface of a flexible substrate in a pattern of discrete reservoirs.When the LLMC contacts an aqueous solution, the silver positiveelectrode (cathode) is reduced while the zinc negative electrode (anode)is oxidized. The LLMC used herein consisted of metals placed inproximity of about 1 mm to each other thus forming a redox couple andgenerating an ideal potential on the order of 1 Volt. The calculatedvalues of the electric field from the LLMC were consistent with themagnitudes that are typically applied (1-10 V/cm) in classicalelectrotaxis experiments, suggesting that cell migration observed withthe bioelectric dressing is likely due to electrotaxis.

Measurement of the potential difference between adjacent zinc and silverdots when the LLMC is in contact with de-ionized water yielded a valueof about 0.2 Volts. Though the potential difference between zinc andsilver dots can be measured, non-intrusive measurement of the electricfield arising from contact between the LLMC and liquid medium wasdifficult. Keratinocyte migration was accelerated by exposure to anAg/Zn LLMC. Replacing the Ag/Zn redox couple with Ag or Zn alone did notreproduce the effect of keratinocyte acceleration.

Exposing keratinocytes to an LLMC for 24 h significantly increased greenfluorescence in the dichlorofluorescein (DCF) assay indicatinggeneration of reactive oxygen species under the effect of the LLMC. Todetermine whether H₂O₂ is generated specifically, keratinocytes werecultured with a LLMC or placebo for 24 h and then loaded with PF6-AM(Peroxyfluor-6 acetoxymethyl ester; an indicator of endogenous H₂O₂).Greater intracellular fluorescence was observed in the LLMCkeratinocytes compared to the cells grown with placebo. Over-expressionof catalase (an enzyme that breaks down H₂O₂) attenuated the increasedmigration triggered by the LLMC. Treating keratinocytes with N-AcetylCysteine (which blocks oxidant-induced signaling) also failed toreproduce the increased migration observed with LLMC. Thus, H₂O₂signaling mediated the increase of keratinocyte migration under theeffect of the electrical stimulus.

External electrical stimulus can up-regulate the TCA (tricarboxylicacid) cycle. The stimulated TCA cycle is then expected to generate moreNADH and FADH₂ to enter into the electron transport chain and elevatethe mitochondrial membrane potential (Δm). Fluorescent dyes JC-1 andTMRM were used to measure mitochondrial membrane potential. JC-1 is alipophilic dye which produces a red fluorescence with high Δm and greenfluorescence when Δm is low. TMRM produces a red fluorescenceproportional to Δm. Treatment of keratinocytes with LLMC for 24 hdemonstrated significantly high red fluorescence with both JC-1 andTMRM, indicating an increase in mitochondrial membrane potential andenergized mitochondria under the effect of the LLMC. As a potentialconsequence of a stimulated TCA cycle, available pyruvate (the primarysubstrate for the TCA cycle) is depleted resulting in an enhanced rateof glycolysis. This can lead to an increase in glucose uptake in orderto push the glycolytic pathway forward. The rate of glucose uptake inHaCaT cells treated with LLMC was examined next. More than two foldenhancement of basal glucose uptake was observed after treatment withLLMC for 24 h as compared to placebo control.

Keratinocyte migration is known to involve phosphorylation of a numberof receptor tyrosine kinases (RTKs). To determine which RTKs areactivated as a result of LLMC, scratch assay was performed onkeratinocytes treated with LLMC or placebo for 24 h. Samples werecollected after 3 h and an antibody array that allows simultaneousassessment of the phosphorylation status of 42 RTKs was used to quantifyRTK phosphorylation. It was determined that LLMC significantly inducesIGF-1R phosphorylation. Sandwich ELISA using an antibody againstphospho-IGF-1R and total IGF-1R verified this determination. As observedwith the RTK array screening, potent induction in phosphorylation ofIGF-1R was observed 3 h post scratch under the influence of LLMC. IGF-1Rinhibitor attenuated the increased keratinocyte migration observed withLLMC treatment.

MBB (monobromobimane) alkylates thiol groups, displacing the bromine andadding a fluorescent tag (lamda emission=478 nm). MCB (monochlorobimane)reacts with only low molecular weight thiols such as glutathione.Fluorescence emission from UV laser-excited keratinocytes loaded witheither MBB or MCB was determined for 30 min. Mean fluorescence collectedfrom 10,000 cells showed a significant shift of MBB fluorescenceemission from cells. No significant change in MCB fluorescence wasobserved, indicating a change in total protein thiol but notglutathione. HaCaT cells were treated with LLMC for 24 h followed by ascratch assay. Integrin expression was observed by immuno-cytochemistryat different time points. Higher integrin expression was observed 6 hpost scratch at the migrating edge.

Consistent with evidence that cell migration requires H₂O₂ sensing, wedetermined that by blocking H₂O₂ signaling by decomposition of H₂O₂ bycatalase or ROS scavenger, N-acetyl cysteine, the increase in LLMC-driven cell migration is prevented. The observation that the LLMCincreases H₂O₂ production is significant because in addition to cellmigration, hydrogen peroxide generated in the wound margin tissue isrequired to recruit neutrophils and other leukocytes to the wound,regulates monocyte function, and VEGF signaling pathway and tissuevascularization. Therefore, external electrical stimulation can be usedas an effective strategy to deliver low levels of hydrogen peroxide overtime to mimic the environment of the healing wound and thus should helpimprove wound outcomes. Another phenomenon observed duringre-epithelialization is increased expression of the integrin subunit αv.There is evidence that integrin, a major extracellular matrix receptor,polarizes in response to applied ES and thus controls directional cellmigration. It may be noted that there are a number of integrin subunits,however we chose integrin αv because of evidence of association of αvintegrin with IGF-1R, modulation of IGF-1 receptor signaling, and ofdriving keratinocyte locomotion. Additionally, integrin_(αv) has beenreported to contain vicinal thiols that provide site for redoxactivation of function of these integrins and therefore the increase inprotein thiols that we observe under the effect of ES may be the drivingforce behind increased integrin mediated cell migration. Other possibleintegrins which may be playing a role in LLMC -induced IGF-1R mediatedkeratinocyte migration are α5 integrin and α6 integrin.

Materials and Methods

Cell culture—Immortalized HaCaT human keratinocytes were grown inDulbecco's low-glucose modified Eagle's medium (Life Technologies,Gaithersburg, Md., U.S.A.) supplemented with 10% fetal bovine serum, 100U/ml penicillin, and 100 λg/ml streptomycin. The cells were maintainedin a standard culture incubator with humidified air containing 5% CO₂ at37° C.

Scratch assay—A cell migration assay was performed using culture inserts(IBIDI®, Verona, Wis.) according to the manufacturers instructions. Cellmigration was measured using time-lapse phase-contrast microscopyfollowing withdrawal of the insert. Images were analyzed using theAxioVision Rel 4.8 software.

N-Acetyl Cysteine Treatment—Cells were pretreated with 5 mM of the thiolantioxidant N-acetylcysteine (Sigma) for 1 h before start of the scratchassay.

IGF-1R inhibition—When applicable, cells were preincubated with 50 nMIGF-1R inhibitor, picropodophyllin (Calbiochem, Mass.) just prior to theScratch Assay.

Cellular H₂O₂ Analysis—To determine intracellular H₂O₂ levels, HaCaTcells were incubated with 5 pM PF6-AM in PBS for 20 min at roomtemperature. After loading, cells were washed twice to remove excess dyeand visualized using a Zeiss Axiovert 200M microscope.

Catalase gene delivery—HaCaT cells were transfected with 2.3×10⁷ pfuAdCatalase or with the empty vector as control in 750 μL of media.Subsequently, 750 μL of additional media was added 4 h later and thecells were incubated for 72 h.

RTK Phosphorylation Assay—Human Phospho-Receptor Tyrosine Kinasephosphorylation was measured using Phospho-RTK Array kit (R & DSystems).

ELISA—Phosphorylated and total IGF-1R were measured using a DuoSet ICELISA kit from R&D Systems.

Determination of Mitochondrial Membrane Potential—Mitochondrial membranepotential was measured in HaCaT cells exposed to the LLMC or placebousing TMRM or JC-1 (MitoProbe JC-1 Assay Kit for Flow Cytometry, LifeTechnologies), per manufacturers instructions for flow cytometry.

Integrin αV Expression—Human HaCaT cells were grown under the MCD orplacebo and harvested 6 h after removing the IBIDI® insert. Staining wasdone using antibody against integrin αV (Abcam, Cambridge, Mass.).

Example 5 Generation of Superoxide

A LLMC system was tested to determine the effects on superoxide levelswhich can activate signal pathways. As seen in FIG. 11, the PROCELLERA®LLMC system increased cellular protein sulfhydryl levels. Further, thePROCELLERA® system increased cellular glucose uptake in humankeratinocytes. Increased glucose uptake can result in greatermitochondrial activity and thus increased glucose utilization, providingmore energy for cellular migration and proliferation. This can speedwound healing.

Example 6 Treatment of a Full-Thickness Wound

A 35-year old male suffers a burn to his shoulder. The burn is excised,then a LLMC system comprising a bioelectric antimicrobial devicecontaining a multi-array matrix of biocompatible microcells is used tocover the wound. The system is designed as described herein to allow formovement about the shoulder joint. The burn heals without the need forskin grafts.

Example 7 Treatment of a Surgical Site

A 56-year old female suffering from squamous cell carcinoma undergoes aprocedure to remove a tumor. The tumor removal site is covered with aLLMC system comprising a bioelectric antimicrobial device containing amulti-array matrix of biocompatible microcells. The surgical site healswith minimal scarring.

Example 8 Treatment of Open Fracture

A 15-year old male suffers a grade-III open tibia-fibula fracture,leaving exposed bone and muscle. The wound is dressed with LLMC systemsas described herein comprising a bioelectric antimicrobial devicecontaining a multi-array matrix of biocompatible microcells. The woundheals without the need of muscle or skin grafts. The wound is also keptfree from microbial contamination as a result of the broad-spectrumantimicrobial effect of the wound management systems as disclosedherein.

Example 9 Treatment of an Insect Bite

A 25-year old male suffers numerous mosquito bites along his legs. ALLEF system including a pliable dressing material as described herein iswrapped around his legs. The LLEF system reduces the swelling andeliminates the itching caused by the bites within 3 hours.

Example 10 Treatment of a Venomous Snake Bite

A 25-year old male suffers a venomous snake bite to his leg. Bleeding isstopped then the wound is dressed with a LLMC system comprising abioelectric antimicrobial dressing containing a multi-array matrix ofbiocompatible microcells. The venom injected during the bite isneutralized. Over the next 2 weeks the wound heals. The wound is alsokept free from microbial contamination as a result of the broad-spectrumantimicrobial effect of the wound management systems disclosed herein.

Example 11 Treatment of Diabetes

A 48-year old woman suffers from type 2 diabetes. To limit glucoseexcursions and lower serum glucose levels, LLEF systems of the inventionare applied around the patient's abdomen and extremities. This increasescellular glucose uptake and reduces serum glucose levels, as well asmoderating glucose excursions.

Example 12 LLMC Influence on Biofilm Properties

In this study ten clinical wound pathogens associated with chronic woundinfections were used for evaluating the anti-biofilm properties of aLLMC. Hydrogel and drip-flow reactor (DFR) biofilm models were employedfor the efficacy evaluation of the wound dressing* in inhibitingbiofilms. Biofilms formed with Acinetobacter baumannii, Corynebacteriumamycolatum, Escherichia coli, Enterobacter aerogenes, Enterococcusfaecalis CI 4413, Klebsiella pneumonia, Pseudomonas aeruginosa, Serratiamarcescens, Staphylococcus aureus, and Streptococcus equi clinicalisolates were evaluated. For antimicrobial susceptibility testing ofbiofilms, 10⁵ CFU/mL bacteria was used in both biofilm models. Forpoloxamer hydrogel model, the LLMCs (25 mm diameter) were applieddirectly onto the bacterial biofilm developed onto 30% poloxamerhydrogel and Muller-Hinton agar plates, and incubated at 37° C. for 24 hto observe any growth inhibition. In the DFR biofilm model, bacteriawere deposited onto polycarbonate membrane as abiotic surface, andsample dressings were applied onto the membrane. The DFR biofilm wasincubated in diluted trypticase soy broth (TSB) at room temperature for72 h. Biofilm formations were evaluated by crystal violet staining underlight microscopy, and anti-biofilm efficacy was assessed by reduction inbacterial numbers. Our data suggests anti-biofilm activity of the LLMCin both biofilm models.

In closing, it is to be understood that although aspects of the presentspecification are highlighted by referring to specific embodiments, oneskilled in the art will readily appreciate that these disclosedembodiments are only illustrative of the principles of the subjectmatter disclosed herein. Therefore, it should be understood that thedisclosed subject matter is in no way limited to a particularmethodology, protocol, and/or reagent, etc., described herein. As such,various modifications or changes to or alternative configurations of thedisclosed subject matter can be made in accordance with the teachingsherein without departing from the spirit of the present specification.Lastly, the terminology used herein is for the purpose of describingparticular embodiments only, and is not intended to limit the scope ofthe present disclosure, which is defined solely by the claims.Accordingly, embodiments of the present disclosure are not limited tothose precisely as shown and described.

Certain embodiments are described herein, including the best mode knownto the inventor for carrying out the methods and devices describedherein. Of course, variations on these described embodiments will becomeapparent to those of ordinary skill in the art upon reading theforegoing description. Accordingly, this disclosure includes allmodifications and equivalents of the subject matter recited in theclaims appended hereto as permitted by applicable law. Moreover, anycombination of the above-described embodiments in all possiblevariations thereof is encompassed by the disclosure unless otherwiseindicated herein or otherwise clearly contradicted by context.

Groupings of alternative embodiments, elements, or steps of the presentdisclosure are not to be construed as limitations. Each group member maybe referred to and claimed individually or in any combination with othergroup members disclosed herein. It is anticipated that one or moremembers of a group may be included in, or deleted from, a group forreasons of convenience and/or patentability. When any such inclusion ordeletion occurs, the specification is deemed to contain the group asmodified thus fulfilling the written description of all Markush groupsused in the appended claims.

Unless otherwise indicated, all numbers expressing a characteristic,item, quantity, parameter, property, term, and so forth used in thepresent specification and claims are to be understood as being modifiedin all instances by the term “about.” As used herein, the term “about”means that the characteristic, item, quantity, parameter, property, orterm so qualified encompasses a range of plus or minus ten percent aboveand below the value of the stated characteristic, item, quantity,parameter, property, or term. Accordingly, unless indicated to thecontrary, the numerical parameters set forth in the specification andattached claims are approximations that may vary. At the very least, andnot as an attempt to limit the application of the doctrine ofequivalents to the scope of the claims, each numerical indication shouldat least be construed in light of the number of reported significantdigits and by applying ordinary rounding techniques. Notwithstandingthat the numerical ranges and values setting forth the broad scope ofthe disclosure are approximations, the numerical ranges and values setforth in the specific examples are reported as precisely as possible.Any numerical range or value, however, inherently contains certainerrors necessarily resulting from the standard deviation found in theirrespective testing measurements. Recitation of numerical ranges ofvalues herein is merely intended to serve as a shorthand method ofreferring individually to each separate numerical value falling withinthe range. Unless otherwise indicated herein, each individual value of anumerical range is incorporated into the present specification as if itwere individually recited herein.

The terms “a,” “an,” “the” and similar referents used in the context ofdescribing the disclosure (especially in the context of the followingclaims) are to be construed to cover both the singular and the plural,unless otherwise indicated herein or clearly contradicted by context.All methods described herein can be performed in any suitable orderunless otherwise indicated herein or otherwise clearly contradicted bycontext. The use of any and all examples, or exemplary language (e.g.,“such as”) provided herein is intended merely to better illuminate thedisclosure and does not pose a limitation on the scope otherwiseclaimed. No language in the present specification should be construed asindicating any non-claimed element essential to the practice ofembodiments disclosed herein.

Specific embodiments disclosed herein may be further limited in theclaims using consisting of or consisting essentially of language. Whenused in the claims, whether as filed or added per amendment, thetransition term “consisting of” excludes any element, step, oringredient not specified in the claims. The transition term “consistingessentially of” limits the scope of a claim to the specified materialsor steps and those that do not materially affect the basic and novelcharacteristic(s). Embodiments of the present disclosure so claimed areinherently or expressly described and enabled herein.

1. A device for reducing inflammation in a wound comprising astretchable substrate comprising biocompatible electrodes capable ofgenerating a low level electric field (LLEF) or low level micro current(LLMC).
 2. The device of claim 1 wherein the biocompatible electrodescomprise a first array comprising a pattern of microcells formed from afirst conductive material, and a second array comprising a pattern ofmicrocells formed from a second conductive material.
 3. The device ofclaim 2 wherein the first conductive material and the second conductivematerial comprise the same material.
 4. The device of claim 3 whereinthe first and second array each comprise a discrete circuit.
 5. Thedevice of claim 4, further comprising a power source.
 6. The device ofclaim 2 wherein the first array and the second array spontaneouslygenerate a LLEF.
 7. The device of claim 6 wherein the first array andthe second array spontaneously generate a LLMC when contacted with anelectrolytic solution.
 8. The device of claim 6 wherein the LLEF isbetween 0.05 and 5 Volts.
 9. The device of claim 8 wherein the LLEF isbetween 0.1 and 5 Volts.
 10. The device of claim 8 wherein the LLEF isbetween 1.0 and 5 Volts.
 11. The device of claim 7 wherein the LLMC isbetween 1 and 200 micro-amperes.
 12. The device of claim 11 wherein theLLMC is between 1 and 100 micro-amperes.
 13. The device of claim 11wherein the LLMC is between 100 and 200 micro-amperes.
 14. The device ofclaim 11 wherein the LLMC is between 150 and 200 micro-amperes.